Methods of determining the effect of an agent on diploid cells and/or on the pattern of expression of polypeptides expressed therewith

ABSTRACT

A method of determining the effect of an agent on a diploid cell and/or on an expression or activity of a polypeptide expressed within the diploid cell is provided. The method is effected by: (a) administering an exogenous RNA molecule encoding the polypeptide into the diploid cell; (b) contacting the diploid cell with the agent; and (c) monitoring a phenotype of the diploid cell and/or the expression or activity of the polypeptide within the diploid cell, thereby determining the effect of the agent on the diploid cell and/or on the expression or activity of the polypeptide expressed within the diploid cell.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of determining the effect of anagent on diploid cells and/or on the pattern of expression ofpolypeptides expressed therewith.

The extensive effort to sequence the human genome as well as the genomeof other vertebrates and invertebrates is expected to revolutionizemedicine and agriculture. To effectively use the libraries of genesequences, the physiological or pathological functions of a given geneor groups of genes has to be understood. This can be achieved by the useof reliable platforms to express the genes and then examine the outcomeof their actions at different levels. The physiological or pathologicaleffects of a gene, or a group of genes, can be studied on the behavioralor morphological levels of a whole animal, system-tissue, cellular orbiochemical levels.

Visualization of the spatiotemporal distribution of the gene's productand their relationship to other proteins, in real time and underdifferent physiological and pathological contexts, is needed in order toanalyze their mechanisms of action and their various functions.Accordingly, chimeric DNA constructs comprising reporter genes [encodingreporter proteins such as green fluorescent protein (GFP) andderivatives (EGFP, YFP, etc.), β-galactosidase, β-glucoronidase, etc.]have been increasingly used in biological research. The reporter genesare typically fused to the genes under study. The fusion constructs areintroduced in to cells, expressed and visualized. Reporter genes have awide variety of applications including visualization of the temporal andspatial distribution of genes expression products at the single celllevel.

Yet, because of technical difficulties such as low transfection rate andpoor spatial-temporal resolution, the use of reporter genes for on linevisualization in differentiated neurons, is not used. Thus, functionaland pathological interactions between gene products and the neuronalenvironment cannot be easily studied.

Difficulties in visualizing gene expression in neutonal cells, are wellillustrated by studies conducted on cultured Aplysia neurons. Thesecells have been extensively used to study neuroplasticity (for reviewsee Kandel 2001), regeneration after trauma (Spira et al., 1993, 1996,1999, 2000), pharmacology, second messenger systems, neuronaldevelopment, synaptogenesis and neuronal network formation (seelitrature cited by Kandel 2001) thereby providing cellular, molecular,biochemical, pharmacological and biophysical background information thatcan be utilized for studying gene functions.

Prior art studies have demonstrated that because of the large size ofthese neurons, localized intracellular changes can be directlyvisualized (Ziv and Spira 1997, 1998; Gabso et al., 1997; Gitler andSpira 1998, 2002). However, all prior attempts to transform Aplysia viaDNA microinjection, including DNA encoding GFP, resulted in poor geneexpression (Chang et al., 2000; DesGroseillers et al., 1987, Kaang etal., 1992; 1993; Kaang 1996a,b; Kim and Kaang 1998; Lee et al., 2000;Martin et al., 1995; Zhao et al., 1994). Therefore, DNA constructsencoding reporter genes such as GFP, have not been useful for studyinggene expression in Aplysia.

Mochida et al (1990), illustrated that injection of mRNA coding fortetanus and botulinum toxins into Aplysia neurons led to down regulationin neurotransmitter release. However, while the pathological effect of(extremely low concentration) butulinum and tetanus toxins wasdetectable (suppressing neurotransmitter release), their expressionlevel was far too low for directly detecting, or visualizing, geneexpression products in situ.

While reducing the present invention to practice, the present inventorsdevised a novel approach which traverses the limitations inherent toprior art methods of studying expression of gene products indifferentiated cells, and in neurons in particular. Thus, the presentinvention provides a useful, convenient, rapid and cost effective toolfor directly visualizing fate and function of gene expression productsin differentiated cells.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided Amethod of determining the effect of an agent on a diploid cell and/or onan expression or activity of a polypeptide expressed within the diploidcell, the method comprising: (a) administering an exogenous RNA moleculeencoding the polypeptide into the diploid cell; (b) contacting thediploid cell with the agent; and (c) monitoring a phenotype of thediploid cell and/or the expression or activity of the polypeptide withinthe diploid cell, thereby determining the effect of the agent on thediploid cell and/or on the expression or activity of the polypeptideexpressed within the diploid cell.

According to further features in preferred embodiments of the inventiondescribed below, the diploid cell is a differentiated cell.

According to still further features in the described preferredembodiments the diploid cell is a neuron.

According to still further features in the described preferredembodiments the administering is effected by microinjection.

According to still further features in the described preferredembodiments the exogenous RNA molecule is a capped messenger RNA.

According to still further features in the described preferredembodiments the polypeptide is conjugated to a detectable label selectedfrom the group consisting of green fluorescent protein (GFP),derivatives of GFP, luciferase, β-glucoronidase, β-galactosidase, andchloramphenicol acetyltransferase.

According to still further features in the described preferredembodiments the monitoring is effected by:

(i) fluorescent microscopy;

(ii) protein expression assay; and/or

(iii) assaying enzymatic activity.

According to still further features in the described preferredembodiments the exogenous RNA molecule encoding the polypeptide is achimeric RNA molecule including a first sequence region encoding thepolypeptide and a second sequence region encoding a reporter molecule,wherein the first and the second sequence regions are linked via aninternal ribosome entry site sequence.

According to still further features in the described preferredembodiments the exogenous RNA molecule encoding the polypeptide is achimeric RNA molecule including a first sequence region encoding thepolypeptide and a second sequence region encoding a reporter molecule,wherein the first and the second sequence regions are in-frame linked.

According to another aspect of the present invention there is provided aneuronal cell comprising a chimeric RNA molecule including a firstsequence region encoding a polypeptide of interest and a second sequenceregion encoding a reporter molecule, wherein the first and the secondsequence regions are linked via an internal ribosome entry sitesequence.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of determining theeffect of an agent on diploid cells and/or on the pattern of expressionpolypeptides expressed therewith.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d are photomicrographs depicting expression of EYFP incultured Aplysia neurons. EYFP was injected into cultured Aplysianeurons 20 hours prior to imaging. FIGS. 1 a and 1 c are differentialinterference contrast images of the neuron. FIGS. 1 c and 1 d areenlargements of the neural segment within the rectangle of FIG. 1 a.FIGS. 1 b and 1 d are confocal images. Note that the fluorescent signalis evenly distributed in the axoplasm. The confocal images were producedusing the following setting: laser intensity 5%; iris setting 1.6; gain100.

FIGS. 2 a-b are photomicrographs depicting EGFP-actin bundles at theleading edge of a growth cone lamellipodium formed following axotomy. AB1 neuron was axotomyzed 28 hours following injection with mRNA encodingthe fusion protein. Transection was followed by rapid extension of thegrowth cone lamellipodium. FIG. 2 a is an image of the growth conelamellipodium taken at the level of the glass substrate 29 min followingaxotomy. FIG. 2 b is an image taken from the same region 3 mm above thesubstrate. Note that in FIG. 2 a, actin puncta are seen along the axonalplasma membrane facing the substrate. The perimeters of the growthcone's lamellipodium contain radially oriented actin bundles. In FIG. 2b, 3 mm above the substrate, the actin polymerise along the axon'splasma membrane. The core of the axoplasm does not contain clear actinnetwork.

FIGS. 3 a-b are photomicrographs depicting the effect of Cytochalasin Bon disassembly of actin bundles at the leading edge of growth cones.Shown are two growth cones formed by a cultured B1 neuron injected withmRNA encoding EGFP-actin fusion protein. FIG. 3 a is a photomicrographshowing the leading edges of the growth cones containing bundles of EGFPlabeled actin. FIG. 3 b is a photomicrograph showing actin bundlesdisassembly and the formation of small actin aggregates within thecollapsed growth cones and the axoplasm, nine minutes following bathapplication of 2 mM cytochalasin B.

FIGS. 4 a-b are photomicrographs depicting the depolymerisation ofmicrotubules within a growth cone formed following axotomy. A culturedB1 neuron was injected with mRNA encoding EGFP-tubulin fusion protein.FIG. 4 a is a photomicrograph depicting EGFP labelled microtubulesradiate from the growth cone centre towards the growth cone'slamellipodium perimeters. FIG. 4 b is a photomicrograph depicting as inFIG. 4 a, only twenty three minutes following bath application of 5 mMnocodosole the microtubules depolymerise.

FIGS. 5 a-d are photomicrographs depicting alterations in thespatiotemporal distribution of EGFP-EB3 following axotomy. FIG. 5 a is aphotomicrograph depicting expression of EGFP-EB3 in the intact axon.FIG. 5 b is a photomicrograph depicting expression of EGFP-EB3 20seconds following axotomy. Note that a transient increase in the freeintracellular calcium concentration was detected parallely (not shown).Further note the pattern of EGFP-EB3 “comet tails”-like fluorescentsignal, associated with the plus end of the microtubules, dissipatingfrom the tip of the transected axon. FIG. 5 c is a photomicrographdepicting expression of EGFP-EB3 following the recovery of the freeintracellular calcium concentration. Note that EGFP-EB3 reassociate withrepolymerizing microtubules. FIG. 5 d is a photomicrograph depictingexpression of EGFP-EB3 10 minutes following axotomy. Note that themicrotubules at the tip of the transected axon undergo additionalchanges that finally lead to the formation of vesicles trap surroundedby microtubules pointing their plus ends to a common center. The Golgiderived vesicles (not shown) were visualized by EGFP-SNAP 25.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of determining the effect of anagent on diploid cells and/or on the pattern of expression ofpolypeptides expressed therewith. Specifically, the present inventioncan be used to identify agents which affect gene expression and functionin neurons, such as the cultured neurons of Aplysia and thus serve as asystem for modeling drug-gene interactions

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

In the post genomic era, an increased amount of genomic information hascreated new challenges for the biological research community and thepharmaceutical industry. These include, functional annotation of yetuncharacterized genes and efficient identification of target genesresponsible for complex disease phenotypes and the use of suchinformation for the development of new and specific classes of drugs.

One approach for obtaining such valuable information is by visualizationof the spatial-temporal distribution of gene products, in real time andunder different physiological and pathological conditions. Accordingly,chimeric DNA constructs including reporter genes such as fluorescentproteins, typically of the green fluorescent protein (GFP) family havebeen increasingly used.

Prior attempts to use such chimeric DNA constructs for real-timevisualization of expression products in differentiated neurons wereunsuccessful, due to very low levels of transcription in these cells anda limited pattern of expression [Kaang (1996a,b); Manseau (2001); Lee(2001)].

Injection of reporter molecules into differentiated neurons has alsobeen attempted. For example, Mochida and co-workers injected mRNA ofvarious toxins into cultured Aplysia neurons. As with intra-cellexpression assays, expression products of micro-injected mRNA could notbe detected visually, rather only functionally, indicating that onlyvery low levels of expression could be achieved using this system[Mochida (1990)]. Other attempts to inject reporter mRNA (i.e., LacZ)into differentiated neuronal cells were unsuccessful [Kaang (1996a,b)].

While reducing the present invention to practice, the present inventoruncovered that direct microinjection of chimeric RNA molecules intodiploid cells e.g., cultured Aplysia neurons, can be used to visualizegene expression in such cells and to study the effect of various agentson gene expression and cell fate.

As is illustrated in the Examples section which follows, microinjectionof capped mRNA constructs of enhanced fluorescent protein linked to agiven gene into the cytosol of cultured Aplysia neurons led, withinhours of injection, to the translation and distribution of thefluorescentlly tagged protein in the differentiated neuron. Thismethodology was employed for the first time to demonstrate structuralevents which take place following axotomy of cultured Aplysia neurons.

Although mRNA injection of chimeric polypeptides into haploid cells(e.g., xenopus oocytes) is routinely practiced, it is well known thatploidy affects gene expression levels, and thus experimentsdemonstrating expression of injected chimeric mRNA in haploid cellscannot be reliably utilized to predict the outcome of such experimentsin diploid cells [van Neck (1992) FEBS Lett. 297:189-195; Galitski(1999) Science 285:251-254].

Thus, the present invention provides a method of determining an effectof an agent on a diploid cell and/or on an expression or activity of apolypeptide expressed within the diploid cell.

As used herein the phrase “diploid cell” refers to a cell which has onechromosome from each parental set. The diploid cell according to thisaspect of the present invention may be of a vertebrate (e.g., human) orinvertebrate (e.g., Aplysia californica) animal origin. Preferably, thediploid cell of the present invention is a differentiated cell.According to a preferred embodiment of the present invention thedifferentiated cell is an Aplysia neuron (See the Background andExamples sections).

The method, according to the present invention is effected byadministering an exogenous RNA molecule to the diploid cell.

As used herein the “exogenous RNA molecule” refers to an RNA molecule ofnaturally occurring nucleotides or analogues thereof which enhancestability and delivery of the exogenous RNA molecule. The exogenous RNAmolecule of the present invention encodes an autologous or heterologouspolypeptide, which localization, activity and/or level of expression aremonitored to determine the effect of the agent thereon and/or on thediploid cell. Typically the exogenous RNA molecule is the mRNA productof in-vitro transcription of a DNA molecule as is further describedhereinbelow.

Prior to, concomitant with or following introduction of the exogenousRNA molecule, the diploid cell is contacted with the agent. Finally, thephenotype of the diploid cell and/or expression or activity of thepolypeptide is monitored to thereby determine the effect of the agent onthe diploid cell and/or on the expression or activity of the polypeptideexpressed within the diploid cell.

As mentioned hereinabove, the exogenous RNA molecule may be used tomonitor the effect of the agent on the diploid cell (e.g., cellmorphology).

Thus., the exogenous RNA molecule may encode a structural protein, suchas actin, or a protein binding thereto (see Examples section whichfollows) or subcellular structure marker such as a cell surface proteinwhich identifies the cell membrane.

Alternatively, the exogenous RNA molecule may encode a polypeptide ofinterest which activity or expression may be studied in response totreatment with the agent.

Preferably, the exogenous RNA molecule, according to this aspect of thepresent invention encodes a chimeric polypeptide which includes thepolypeptide of interest fused in frame to a detectable polypeptide. Itwill be appreciated, however, that although the nature of the detectablepolypeptide is of no significance, it should not alter the threedimensional structure of the polypeptide of interest in such fusions.

Alternatively, the chimeric mRNA can include an out-of-frame fusion ofthe two coding sequences encoding the polypeptide of interest and thedetectable polypeptide provided that the downstream coding sequences ispreceded by an internal ribosome entry site (IRES). IRES elements areable to bypass the ribosome scanning model of 5′ methylated Capdependent translation and begin translation at internal sites [Pelletierand Sonenberg, (1988) Nature. 334(6180):320-5]. A number of IRESelements are known in the art such as, for example, the IRES elements ofthe picanovirus family (polio and encephalomyocarditis), which have beendescribed by Pelletier and Sonenberg, (1988) supra, as well an IRES froma mammalian message [Macejak and Sarnow, (1991) Nature. 353(6339):90-4].When the IRES element is present on an mRNA downstream of atranslational stop codon, it directs ribosomal re-entry [Ghattas et al(1991) Mol. Cell. Biol. 11:5848-5959], which permits initiation oftranslation at the start of a second open reading frame). In thismanner, multiple open reading frames can be transcribed together, eachseparated by an IRES, creating polycistronic messages. By virtue of theIRES element, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

A number of polycistronic vectors are known in the art, which may beused in the present invention [see de Felipe (2002) Curr. Gene Ther.2(3):355-78; Vagner (2001) EMBO Rep. 2(10):893-8]. For example, thebicistronic expression plasmid, pIRES1neo which is available fromClontech, Palo Alto, Calif. contains the human cytomegalovirus (CMV)major immediate early protein/enhancer followed by a multiple cloningsite (MCS); a synthetic intron; and the encephalomyocarditis virusinternal ribosome entry site (IRES), followed by the neomycinphosphotransferase gene, with a downstream bovine growth hormonepolyadenylation signal.

As used herein the phrase detectable polypeptide refers to apolypeptide, which can be detected directly or indirectly. For example,the detectable polypeptide can be a fluorescer such as the polypeptidesbelonging to the green fluorescent protein family including the greenfluorescent protein (GFP), the yellow fluorescent protein (YFP), thecyan fluorescent protein (CFP) and the red fluorescent protein (RFP) aswell as their enhanced derivatives. In such a case, the detectablepolypeptide can be detected via its fluorescence, which is generatedupon the application of a suitable excitatory light. The detectablepolypeptide can also be an enzyme which when in the presence of asuitable substrate generates chromogenic products. Such enzymes includebut are not limited to alkaline phosphatase, β-galactosidase,β-D-glucoronidase (GUS) and the like. Alternatively, the detectablepolypeptide can be an epitope tag, a fairly unique polypeptide sequenceto which a specific antibody can bind without substantially crossreacting with other cellular epitopes. Such epitope tags include a Myctag, a Flag tag, a His tag, a Leucine tag, an IgG tag, a streptavidintag and the like. Further detail of polypeptide labels can be found inMisawa et al.

A number of methods for generating and purifying RNA molecules are knownin the art and described in the Materials and Experimental Proceduressection of the Examples section, which follows and in Sambrook, Fritsch,Maniatis (1989). Molecular Cloning: A laboratory manual. CSH LaboratoryPress

For example the exogenous RNA moclecules of the present invention may begenerated by in vitro transcription. In vitro transcription is theprocess by which RNA polymerase, in the presence of purified reactioncomponents, mimics in vivo transcription and directs the generation ofan RNA transcript from a DNA template.

Thus, a DNA polynucleotide encoding the exogenous RNA molecule of thepresent invention is ligated into a nucleic acid vector. A number ofvectors designed for in-vitro transcription as well as cloning purposesare known in the art and may be commercially obtained (seewww.promega.com/vectors/).

It will be appreciated that high quality DNA is required in order toachieve high yield (i.e., above 0.7 mg/ml) of in-vitro generated RNAtranscripts. Template DNA may be linear or circular, includingsupercoiled. Measures are taken that the supercoiled plasmid contain anRNA polymerase termination signal to avoid rolling circle transcription.Rolling circle transcription will produce a larger than expected RNAtranscript when the reaction products are resolved on a denaturingagarose gel. To eliminate this problem, linearizing the template with arestriction enzyme that leaves either a blunt end or a 5′-overhang ispreferably effected. Preferably linearizing templates with a restrictionenzyme that leaves a 3 ′-overhang is avoided, as RNA polymerases mayinitiate transcription on the overhang, producing end-to-endtranscripts.

It will be appreciated that PCR products can also be used as templatesby including an RNA promoter sequence at the 5′ end of eitheramplification primer. These bases become double-stranded promotersequences during PCR.

Typically used RNA polymerases are SP6, T7 and T3 polymerases. These RNApolymerases are DNA template-dependent and have distinct, highlyspecific promoter sequence requirements. Following binding of the of theRNA polymerase to the promoter thereof, the enzyme separates the two DNAstrands and uses the 3′>5′ strand as the template for the synthesis of acomplementary 5′>3′ RNA strand. Depending on the orientation of the DNAsequence relative to the promoter, the template is designed to generatesense or anti-sense strand RNA.

The DNA template contains a double-stranded promoter region where thepolymerase binds and initiates RNA synthesis. Typically usedtranscription templates are plasmids, which contain two unique RNApolymerase promoters, that flank the multiple cloning site and thusallow transcription of either strand of an inserted sequence.

To enhance mRNA processing, stability and nucleocytoplasmic transport invivo, the exogenous RNA molecules of the present invention are capped atthe 5′ end. Substitution of Cap analogue for a portion of the GTPpresent in an in vitro transcription reaction will result in thesynthesis of transcripts with a cap on the 5′-end of the RNA.

Once generated and preferably purified the exogenous RNA molecule isintroduced into the diploid cell. A number of methods for introducingRNA molecules into cells are known in the art. Examples include but arenot limited to transfection and microinjection.

A number of transfecting agents for introducing mRNA molecules are knownin the art [see Bettinger (2001) Curr. Opin. Mol. Ther. 3:116-124].Examples include but are not limited to DEAE-dextran [Malone (1989)Proc. Natl. Acad. Sci. USA 86:6077-81], poly(L-lysine) [Fisher (1997)Biochem. J. 321:49-58], dendrimers [Strobel (2000) Gene Ther. 7:2028-35]and DOTAP lipoplexes [Bettinger (2001) Nucleic Acids Res. 29:3882-91].

Preferably, introduction of the exogenous RNA molecule, according to thepresent invention is effected by microinjection (see Examples section),since this procedure allows cellular introduction of large RNAmolecules. Microinjection is the loading or transfer of a dissolvedsubstance (e.g., RNA) into a living cell. Typically, the tip of a glassmicrocapillary has an inner diameter between 0.2 and 1 μm The capillaryis back loaded with the RNA to be transferred into the cells culturedfor microinjection.

To visualize and evaluate the success of a microinjection procedure, RNAis typically mixed with dyes or labeled with fluorescent markers such asflourescein or rhodamine. The capillary pricks the cell, and RNA(approximately 10% of the cell volume) is transferred from the capillaryinto the cell due to pressure exerted on the capillary via themicroinjector. Preferred concentration for RNA injection, according tothis aspect of the present invention is a volume of about 10% of thecell body at a source concentration of 2-3 μg/μl. Preferred embodimentsare described in the Materials and Experimental Procedures section ofthe Examples section.

As mentioned hereinabove, the diploid cell may be contacted with theagent, prior to, concomitant with or following introduction of the RNAmolecule.

As used herein, the term “agent” refers to a molecule or a condition.Examples of molecules which can be utilized as agents according to thepresent invention include, but are not limited to, nucleic acids, e.g.,polynucleotides, ribozymes, and antisense molecules (including withoutlimitation RNA, DNA, RNA/DNA hybrids, peptide nucleic acids, andpolynucleotide analogs having altered backbone and/or bass structures orother chemical modifications); proteins, polypeptides, carbohydrates,lipids and “small molecule” drug candidates. “Small molecules” can be,for example, naturally occurring compounds (e.g., compounds derived fromplant extracts, microbial broths, and the like) or synthetic organic ororganometallic compounds having molecular weights of less than about10,000 daltons, preferably less than about 5,000 daltons, and mostpreferably less than about 1,500 daltons.

Examples of conditions suitable for use as agents according to thepresent invention include, but are not limited to culturing conditions,such as, for example, temperature, humidity, atmospheric pressure, gasconcentrations, growth media, contact surfaces, radiation exposure (suchas, gamma radiation, UV radiation, X-radiation), injury (e.g., axotomy)and the presence or absence of other cells in a culture.

The agent can be either contacted with or introduced into the cell,using molecular or biochemical methodologies well known in the art.Examples include but are not limited to, transfection, conjugation,electroporation, calcium phosphate-precipitation, direct microinjection,liposome fusion and the like. Selection of a suitable introductionmethod is dependent upon the host cell and the type of agent used.

Once the exogenous RNA molecule is expressed (i.e., translated),typically between 2-24 hours following introduction, the effect of theagent may be monitored. It will be appreciated that in contrast to DNA,RNA administration directs very rapid expression of the encodedpolypeptide thus allowing determination of the studied effect withinminutes of RNA administration.

As mentioned hereinabove, monitoring of the phenotype of the diploidcell and/or the expression or activity of the polypeptide within thediploid cell may be effected using fluorescent microscopy. Directfluorescent microscopy may be applied when the polypeptide includes thedetectable portion e.g., GFP, as described above. Alternatively,detection may be effected using fluorescently-labeled antibodies whichbind directly or indirectly the polypeptide and/or an epitope tagconjugated thereto.

Alternatively, monitoring is effected by assaying enzymatic activity ofthe polypeptide or the detectable label, as described above. Examplesinclude but are not limited to kinase activity, phosphatase activity,lipase activity, galacto/glucosidase activity and the like.

Alternatively, the agent may affect the level of expression of theencoded polypeptide. In this case monitoring may be effected usingprotein expression assays which are well known in the art such asWestern blotting and staining.

It will be appreciated that when the normal phenotypic pattern (e.g.,level of expression, cellular distribution, biochemical modification,activity etc.) of the polypeptide within the diploid cell is known, sucha normal pattern can be used to identify agents which have an effect onthe diploid cell and/or on the expression or activity of the polypeptideexpressed within the cell.

Alternatively, determination of the effect of the agent on the diploidcell and/or on the expression or activity of the polypeptide expressedwithin the cell is effected by comparing the pattern (i.e. activity,level and localization) of expression of the polypeptide, followingagent treatment, with a similar manipulated cell, which was not treatedwith the agent.

Still alternatively, the effect of the agent may be determined bycomparing the pattern of expression of the polypeptide, prior to, andfollowing agent treatment.

Although the present invention is practiced with a single cell, such amethod is preferably used for high throughput screening of agents usinga plurality of cells to simultaneously screen a variety of agents. Whena large number of cells are microscopically scanned, an automatic highthroughput screening is effected using a microscope combined with adigital camera and any one of a number of pattern recognitionalgorithms, such as the product distributed under the commercial nameARAYSCAN by Cellomics Inc., U.S.A

Thus, in one example, cells are distributed into flat glass-bottommultiwell (96) plates at a precalibrated density that allows the growthof just one or two clones per well. In a typical experiment, between10-100 plates are prepared and examined microscopically. This screen canbe carried out manually. However, it is possible to install an automatedstage, for example multiwell attachment for the DeltaVision microscope,Cellomics automated microscope, or an equivalent.

Once identified, agents having an effect on a diploid cell and/or onexpression or activity of the polypeptide expressed therewith arepreferably recovered.

The retrieved agents are further analyzed for their exact mechanism ofaction and adjusted for optimal effect, using various biochemical andcell-biology methods. Eventually, distinguishing which of the agentisolated is a potential lead compound can be accomplished by testing theeffect of the agent in pharmacological models of various diseases.Agents that affect disease progression or onset, constitute leads fordrug development.

In summary, the present invention provides a novel approach forvisualizing fate and function of gene expression products within cells,preferably differentiated cells, most preferably neurons. Morespecifically, the present invention may provide tools to facilitateresearch on, for example, expression and function of genes;spatiotemporal distribution of gene products; intracellular interactionsbetween genes and gene products; effect of drugs, bioactive materials,neurotransmitters and modulators, electrical activity and manipulationthat mimic neurotrauma on gene expression, distribution and function;and analyzing role of transcription factors, membrane properties, signaltransduction, growth, regeneration, learning and memory. Hence, theinvention provides a useful tool for monitoring expression, distributionand function of genes within cells, that is efficient, sensitive,selective, rapid, convenient, and cost effective.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in. Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Materials and Experimental Procedures

Solutions—L-15 supplemented for marine species (msL-15) was prepared aspreviously described [Schacher and Proshansky (1983)]. Briefly,Leibovitz's L-15 Medium (Gibco-BRL, Paisley, Scotland) was supplementedwith 12.5 g/L NaCl, 6.86 g/L D(+) Glucose.H₂O, 3.15 g/L anhydrous MgSO₄,344 mg/L KCl, 192 mg/L NaHCO₃, 5.7 g/L MgCl₂.6H₂O and 1.49 g/LCaCl₂.2H₂O. Penicillin, streptomycin and amphotericin B (BiologicalIndustries, Kibbutz Beit Haemek, Israel) were added up to finalconcentrations of 100 units/ml, 0.1 mg/ml and 0.25 μg/ml respectively.

Culture medium included 5-20% filtered hemolymph obtained from Aplysiafaciata (specimens were collected along the Mediterranean coast) dilutedin ms-L 15.

Artificial Sea Water (ASW) included NaCl 460 mM, KCl 10 mM, CaCl₂ 10 mM,MgCl₂ 55 mM, HEPES 10 mM, adjusted to pH 7.6.

Cell culture—Neurons B1 and B2 from buccal ganglia, MCn neurons from themetacerebral ganglion, sensory neurons from the pleuropeadal ganglionand LUQs from the abdominal ganglion of Aplysia californica wereisolated and maintained in culture as previously described [Schacher andProshansky, (1983); Spira et al., (1993, 1996)].

Briefly, juvenile Aplysia californica (1-10 gr) were anesthetized byinjection of isotonic MgCl₂ solution (380 mM) into the animal's bodycavity.

Buccal ganglia were dissected and incubated in ms-L15 containing 1%protease (type IX, Sigma, Rehovot, Israel) at 34° C. for 1.5-2.5 h.Following the protease treatment the ganglia were washed with ms-L15,pinned and desheated. The identified neurons were manually pulled outalong with their original axon with the aid of a sharp glassmicroelectrode. The neurons were immediately plated in glass-bottomdishes coated with poly-L-lysine (Sigma, Rehovot, Israel) containingculture medium.

All microinjections were performed 8-24 hours from plating, at roomtemperature (21-25° C.) after replacing the culture medium with ASW.

Expression constructs—Enzymes were obtained from (New England Biolabs,Beverly, Mass., USA). Each of the chimeric cDNAs of EGFP-EB3, EGFP-p50and EGFP-MLIV was subcloned into the pCS2+ expression vector asdescribed below [Rupp (1994); Turner and Weintraub (1994)]. For EGFP-EBthe pCS2+ vector was cut with XhoI and SnaB. The insert (EGFP-EB3) wascut with NotI, fill in and cut with SalI. For EGFP-P50 the pCS2+ vectorwas cut with XbaI and the insert was cut with NheI and SpeI.

EGFP cDNA was amplified from pEGFP-N1 vector (Clontech, Palo-Alto,Calif., USA) by polymerase chain reaction (PCR) with two specificprimers: 5′-GGCCATGGTGAGCAAGG-3′ and 5′-CTTGTACAGCTCGTCCATG-3′(GensetOligos) (SEQ ID NOs: 1 and 2, respectively). The PCR product wasdigested with HindII and SmaI and subcloned into corresponding sites ofBluescript II SK (Stratagen, La Jolla, Calif.). Aplysia actin providedby Dr DesGrosiller (Montreal University, Canada) was amplified by PCRfrom Bluescript containing the actin cDNA using two specific primers:5′-ATGTGTGACGACGATGTT-3′ and 5′-TTAGAAGCACTTGCGGTCG-3′ (SEQ ID NOs: 3and 4, respectively) with SmaI and XbaI restriction sites at their 5′ends. Following digestion, the PCR product was subcloned in-frame withEGFP into the previously prepared pBluescript-EGFP vector linearizedwith SmaI and XbaI. The EGFP-Actin fragment was then cut out frompBluescript with ClaI and XbaI and subcloned into corresponding sites ofpCS2+ vector [Rupp (1994); Turner and Weintraub (1994) supra].

EYFP, EBFP, ECFP and RFP constructs were prepared as described forEGFP-actin, hereinabove. EYFP, EBFP and ECFP cDNAs were amplified frompEYFP, pEBFP and pECFP, respectively (Clontech) by PCR using the primersset forth in SEQ ID NOs: 1 and 2. The RFP cDNA was amplified frompDsRed1-N1 vector (Clontech) using the following primers:5′-GGCCACCATGGTGCGCTCCT-3′ and 5′-CAGGAACAGGTGGTGGCGG-3′(Genset Oligos,SEQ ID NOs: 5 and 6, respectively).

Aplysia SNAP-25 provided by Dr W. Sossin, University of Montreal Canadawas amplified by PCR using the following SmaI and SpeI restriction sitescontaining primers: 5′ GTCCCCCGGGATGGCGGCGCCAGCGGAG 3′ and 5′GCGGACTAGTCTAAGCCTCCTTAAGCAG 3′ (SEQ ID NOs: 7 and 8, respectively). Theresultant digested PCR product was used to replace the actin gene fromthe pCS2-EGFP-actin that was excised by SmaI and XbaI.

PCS2-EGFP-α-tubulin was prepared from Clontech pEGFP-Tub (Cat. #6117-1).This plasmid encodes a fusion protein including EGFP and humanα-tubulin. The plasmid was digested with BamHI, filled in with T4 DNApolymerase, digested with NheI and ligated to PCS2 cut by XbaI andSnaBI.

In-vitro transcription—5′-capped and 3′-polyadenylated mRNA was in vitrotranscribed using recombinant in vitro transcription system (Promega,Madison, Wis., USA,). 10 μg of NotI-linearized pCS2+ was used as atemplate to transcribe capped mRNA. The transcription reaction waseffected with RiboMax-sp6 kit (Promrga-P1280). Briefly, a reactionmixture was prepared by mixing 8 μl Transcription ×5 Buffer, 8 μl rNTPsmix containing 25 mM CTP, ATP, UTP and 12 mM GTP, 4 μl of 15 mM Capanalog (Roche 85846029), 1 μl of 40 units rRnasin (Promega N251A), 4 μlenzyme mix and 1-2 μg linear plasmid. Final reaction volume wascompensated to 40 μl. Reaction was incubated for 2-4 hours in 37° C. RNAwas purified by Rneazy mini kit (Qiagene, Cat. No. 74104) and the cleanRNA was eluted to a final volume of 25-40 μl and stored at −80° C. Theconcentration of RNA used for injection did not exceed 5 μg/μl.

mRNA microinjection—mRNA was injected into the cytoplasm of Aplysianeurons bathed in ASW 8-48 hours following plating. 0.5-5 μg/μl mRNA in80 mM KCl was used for injection. Injection was performed by pressureusing Medical System Corp microinjector inserted into the cell bodyunder visual control. Approximately up to 10% of the cell's body volumewas injected. The same micropipette used for injection was also used tocontinuously monitor the transmembrane potential and input resistance[Benbassat and Spira (1993) Exp. Neurol. 122:295-310]. Good penetrationwas indicated by a resting potential of >−35 mV and measuring a typicalinput resistance. At the end of the injection, the micropipette tip waspooled out gently of the neuron. Cells were imaged for proteinexpression 12-48 hours following injection.

Mag-fura-2 Ca²⁺ imaging—To study the local effect of elevated calciumlevels on the distribution of a given fluorescentlly labled proteinratio imaging of the free intracellular calcium concentration waseffected by mag fura-2. Mag-fura-2 loading, imaging and calibration wasdone as previously described [Ziv and Spira, (1993, 1995, 1997)]. Thefluorescence microscopy system consisted of a Zeiss Axiovert microscopeequipped with a 75 W Xenon arc lamp, a Zeiss 40×0.75 NA Plan-Neofluarobjective, 340±5 nm and 380±5 nm bandpass excitation filters set in acomputer-controlled, Lambda10 position filter changer (Sutter, NovatoCalif.), a dichroic mirror with a cut-off threshold of 505 nm and a545±25 nm band pass emission filter. The images were collected with anintensified CCD video camera (Hamamatsu, Japan), stored as computerfiles and processed using a software package written in our laboratory.

Proteolytic activity imaging—The effect of activated calpain on thebehavior of fluorescentlly labeled protein was imaged on line calpainactivity. Imaging of proteolytic activity was performed as previouslydescribed [Gitler and Spira, (1998, 2002)]. Neurons which werepreviously loaded with mag-fura-2 were continuously incubated in ASWcontaining 10 μM bis(CBZ-Alanyl-Alanine amine) Rhodamine 110 (bCAA-R110,Molecular Probes, Eugene Oreg.) and were imaged for the production offluorescent Rhodamine 110 (R110). Ratio imaging was used to correct forvolumetric changes, and was performed as described for mag-fura-2 exceptthat the excitation wavelengths used were 490±6 nm, which excites R110,and 350±5 nm, which is the isosbestic point of mag-fura-2.

Axotomy—Axonal transection was performed by applying pressure on theaxon with the thin shaft of a micropipette under visual control, aspreviously described [Spira et al., 1993, 1996; Ziv and Spira, (1993)].

Example 1 Expression of EGFP and EYFP in Aplysia Neurons

The ability of mRNA injection to direct protein expression in neuronalcells was addressed in cultured Aplysia neurons.

Results

In vitro transcribed mRNA encoding EGFP or EYFP was injected intocultured Aplysia neuron. Cells were microscopically examined 12-24 hoursfollowing manipulation. As shown in FIGS. 1 a-b, EGFP and EYFPexpression was observed in about 100% of the injected neurons.Expression was observed in the cell-body, the axons and the neuritis andthe fluorescent signal was evenly distributed in the cytoplasm.

Example 2 EGFP-Actin Expression in Cultured Aplysia Neurons

The translational efficiency of mRNAs encoding EGFP-tagged actin andtubulin was examined in cultured Aplysia neurons.

Results

As shown in FIGS. 2 a-b, injection of a solution containing mRNAencoding EGFP-actin fusion protein resulted in a high fluorescent signalin the cell body, axons and neuritis. The fluorescent signal appeared tobe distributed homogeneously in the cytoplasm of the cell body, mainaxon and neurites but was not detected within the nucleus. Fluorescenthot spots, possibly representing adhesion plaques, were seen along theplasma membrane facing the substrate.

To determine whether the observed fluorescent signal corresponded toEGFP-tagged actin, rather than to EGFP alone, the main axon wastransected and fluorescent signal distribution was imaged during theformation and extension of the lamellipodium of the growth cone. Aspreviously described, axonal transection of cultured Aplysia neuronleads to the rapid formation of a growth cone lamellipodium at the tipof the cut axon [Ziv and Spira, (1995); Ashery et al., (1996); Gitlerand Spira, (1998, 2002); Spira et al., (2001)]. It is also welldocumented that the growth cone's lamellipodium perimeters are rich withactin filaments that are central to its motility (Forscher and Smith,1988; Lin et al., 1994; Tanaka and Sabry, 1995; Scheafer et al., 2002).

Indeed as shown in FIGS. 2 a-b, following axotomy of manipulatedneurons, a growth cone in the form of large lamellipodium was evidentwithin 10_(—)/15 min of axotomy. The perimeters of the growth cone wererich with fluorescent actin bundles while the central region exhibitedfluorescent puncta (FIGS. 2 a-b). As shown in FIGS. 3 a-b, thefluorescent bundles depolymerised within minutes of cytochalasin Bapplication.

Thus, it is concluded that these bundles were EGFP labelled actinbundles. These results demonstrate that the fluorescent signalcorresponds to expressed EGFP-actin fusion protein, which incorporatesinto the actin skeleton and allows the visualisation of the dynamicbehaviour of this skeletal component.

Example 3 Expression of EGFP-Tagged Tubulin in Cultured Aplysia Neurons

To establish that the above-described methodology can be used as areliable tool to express various types of proteins in cultured Aplysianeurons, the mRNA of EGFP-tagged tubulin was injected into culturedAplysia neurons.

Results

On-line confocal microscope imaging of neurons injected by a solutioncontaining mRNA encoding EGFP-a tubulin fusion protein resulted inincorporation of the tagged tubulin into microtubules that extended intoan axotomy induced growth cone's lamellipodium (FIG. 4 a). Bathapplication of the microtubules depolymerizing agent nocodazole (5 mM)for 5 min resulted in depolymerisation of most microtubules (FIG. 4 b).

Example 4 Expression of EYFP-Tagged SNAP-25 in Cultured Aplysia Neurons

Similarly to Example 3, above, SNAP-25 detectable protein was injectedinto cultured Aplysia neuroms. SNAP-25 is a member of the SNARE complex,the synaptosome associated protein of 25 kDa (SNAP-25).

The fluorescent signal of SNAP-25 revealed the presence of fluorescentspots in the intact axon as well as in the growth cone (not shown).

Example 5 Expression of the End Binding Protein 3 (EB3) in AplysiaNeurons

Background

End Binding Protein 1 (EB1), is a protein known to bind to APC(adenomatus polyposis coli tumor suppressor gene) which depletescytoplamic β-catenins. EB1 associate with MTs of the mitotic spindle andis important in spindle assembly throughout the cell cycle. End BindingProtein 3 (EB3) a homologue of EB1, was recently isolated from humanfetal brain [Nakagawa et al., (2000)]. The full-length cDNA of EB3encodes a protein of 282 amino acids with 54% identity to EB1 but isexpressed preferentially in brain tissue. EB3 binds to APCL which isthought to play a role in differentiation of the nervous system. Theconservation of the armadillo domain by APC and APCL (76% identity)suggests that both interact with similar proteins. APCL can interactwith β-catenin, and deplete intracellular β-catenin as APC does.

To shed light on the function of EB3 in neuronal cells, a GFP-EB3 mRNAwas injected into cultured Aplysia neurons and protein expression,localization and activity was determined.

Results

As shown in FIG. 5 a, GFP-EB3 was expressed within 5 hr of mRNAinjection into cultured Aplysia neurons. GFP-EB3 was able to bind instretches to the plus end of microtubules, move with the growing MT'stips to thereby form a comet tail-like structure.

Thus, the expression of human GFP-EB3, in cultured Aplysia neuronsenabled to probe on line the polarity and dynamics of MTs in theneurons, the dynamic of microtubules organizing center, the role of MTsin GCs formation, neurites extension and the effect of drugs on MTs.

Example 6 Imaging cellular Cascades Using EGFP-EB3-Expressing AplysiaNeurons

Background

To illustrate the utility of cultured Aplysia neurons as an expressionplatform for mammalian genes, several parameters which are involved inthe cellular cascade leading to the formation of a growth cone followingaxotomy were imaged. Such a cascade is initiated by a transient andlocal elevation of the free intracellular Ca²⁺ concentration([Ca²⁺]_(i)), which is followed by localized activation of the calciumdependent protease calpain. Calpairi-dependent proteolysis leads torestructuring of the microtubules (MTs) and neurofilaments to form aspecialized cytoskeletal compartment that “traps” and “hold” Golgiderived transported vesicles. The “vesicles trap” is formed withinseconds of axotomy 100-150 μm posterior to the site of transection.

Results

Confocal microscope imaging of EGFP-EB3 throughout the process revealedthat during the elevation of the [Ca²⁺]_(i) the MTs undergo two cyclesof depolymerizations (FIG. 5 b) and repolymerizations (FIG. 5 c).Thereafter, the MTs repolymerize to form the vesicles “trap” by thereorientation of MTs plus ends into a common center (FIG. 5 d).

Local elevation of the [Ca²⁺]_(i) by ionomycin application was able tomimic the process, demonstrating that the transient elevation in the[Ca²⁺]_(i) rather than other injury related events underlie the process.In addition to that it was also observed that inhibition of calpain bycalpeptin induces milled dissociation of EB3 from the MTs. Nacodasolinduces MTs deplymerization and dissociation of EB3 from the MTs.

Example 7 Expression of EGFP-Dynamitin (P50) Construct

Through interactions with organelle-bound MT motors translocateorganelles. The Kinesin family motors translocate organelles toward theplus end (cell periphery) and dynein translocate toward the minus end(cell center). Dynactin is a multisubunit complex that plays anaccessory role in cytoplasmic dynein function. P50 (GenBank AccessionNo. AF200744), is a subunit of the Dynactin complex. Focusing of theminus ends into radial array is generally related to MTs outgrowth fromthe centrosom. Nevertheless, dynein forms complexes that are capable ofinteracting with more than one MT. Since under these conditions dyneinremains attached to the minus end (in centrosom free conditions and thepresences of active dynein) a self-organization condition is formedwhich drives the MTs to form a focused array. GFP-p50 was expressedwithin 5 hr. of mRNA injection into cultured Aplysia neurons.

Example 8 Expression of ML VIV (Mucolipidosis Type IV)

Mucolipidosis type VI is a neurodegenerative lysosomal storage disordercharacterized by psychomotor retardation. The diseases is classified asmucolipidosys due to the simultaneous lysisosomal storage of lipids andwater soluble substrate. The MLVIV gene is involved in regulation of theendocytotic pathway (Bach 2001). GFP-MLVIV was expressed within 5 hr. ofmRNA injection into cultured Aplysia neurons.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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1. A method of determining the effect of an agent on a diploid celland/or on an expression or activity of a polypeptide expressed withinthe diploid cell, the method comprising: (a) administering an exogenousRNA molecule encoding the polypeptide into the diploid cell; (b)contacting the diploid cell with the agent; and (c) monitoring aphenotype of the diploid cell and/or the expression or activity of thepolypeptide within the diploid cell, thereby determining the effect ofthe agent on the diploid cell and/or on the expression or activity ofthe polypeptide expressed within the diploid cell.
 2. The method ofclaim 1, wherein the diploid cell is a differentiated cell.
 3. Themethod of claim 2, wherein the diploid cell is a neuron.
 4. The methodof claim 1, wherein said administering is effected by microinjection. 5.The method of claim 1, wherein said exogenous RNA molecule is a cappedmessenger RNA.
 6. The method of claim 1, wherein the polypeptide isconjugated to a detectable label selected from the group consisting ofgreen fluorescent protein (GFP), derivatives of GFP, luciferase,β-glucoronidase, β-galactosidase, and chloramphenicol acetyltransferase.7. The method of claim 1, wherein said monitoring is effected by: (i)fluorescent microscopy; (ii) protein expression assay; and/or (iii)assaying enzymatic activity.
 8. The method of claim 1, wherein saidexogenous RNA molecule encoding the polypeptide is a chimeric RNAmolecule including a first sequence region encoding the polypeptide anda second sequence region encoding a reporter molecule, wherein saidfirst and said second sequence regions are linked via an internalribosome entry site sequence.
 9. The method of claim 1, wherein saidexogenous RNA molecule encoding the polypeptide is a chimeric RNAmolecule including a first sequence region encoding the polypeptide anda second sequence region encoding a reporter molecule, wherein saidfirst and said second sequence regions are in-frame linked.
 10. Aneuronal cell comprising a chimeric RNA molecule including a firstsequence region encoding a polypeptide of interest and a second sequenceregion encoding a reporter molecule, wherein said first and said secondsequence regions are linked via an internal ribosome entry sitesequence.